Download Isolation of a New, Pink, Obligately Thermophilic, Gram

INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,
OCt. 1975, p. 357-364
Copyright 0 1975 International Association of Microbiological Societies
Vol. 25, No. 4
Printed in U.S.A.
Isolation of a New, Pink, Obligately Thermophilic, GramNegative Bacterium (K-2 Isolate)
ROBERT F. RAMALEY, KEITH BITZINGER,’ RICHARD M. CARROLL, AND RICHARD B. WILSON
Departments of Biochemistry and Pathology, University of Nebraska College of Medicine, Omaha,
Nebraska M105
A pink, obligately themophilic (60 C ) , gram-negative, nonmotile, nonsporeforming, rod-shaped bacterium with a deoxyribonucleic acid base ratio of 64 to 65
mol% guanine plus cytosine has been repeatedly isolated from slightly alkaline,
man-made and natural thermal aquatic environments. This bacterium (K-2
isolate) does not appear to have been described previously and it has been
deposited with the American Type Culture Collection as an unidentified bacterium with the accession no. 27599.
Prior to 1969, the bacterium with the highest
known optimum growth temperature in laboratory culture was Bacillus stearothermophilus,
and the majority of the work on “thermostable”
enzymes and other subcellular components was
carried out with this gram-positive bacterium
(15). However, in 1969, Brock and Freeze (3)
reported the isolation of a gram-negative, obligately thermophilic bacterium, Thermus aquaticus, that had an optimum growth temperature of 70 C . Since that time, additional gramnegative thermophilic isolates have been obtained that also have optimum growth temperatures of 70 C , including Thermus X-l(14),Thermus thermophilus (13), and Thermomicrobium
roseum (10).
The present report describes the physiologic
and morphologic properties of a pink, gramnegative, obligately thermophilic bacterium
that has a slightly lower “optimum” growth
temperature (60 C) but that might eventually
be classified as a species of Thermus.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The
“original” K-2 isolate was obtained from a thermally
polluted stream on the Indiana University campus
in Bloomington, Indiana, as described in the Results
section of this paper. This isolate has been deposited
in the American Type Culture Collection (ATCC),
Rockville, Md., as a pink, obligately thermophilic,
unidentified bacterium (K-2 isolate) (ATCC accession no. 27599). Isolate P-P-1 was obtained from the
effluent channel of natural hot springs (Poncha
Springs) (7, 18) near Poncha City, Colo., in July 1973.
Isolates R-P-1 and R-P-2 were obtained from the
effluent channel of natural hot springs (Routt Hot
Springs) (7, 18) 7 miles (ca. 11.3 km) north of Steamboat Springs, Colo., in August 1973. Thermus aquaticus YT-1(3) was obtained from T. D. Brock (Depart-
’ Present address: Miles Laboratories, Elkart, Ind.
46514.
ment of Microbiology, University of Wisconsin, Madison). Bacillus stearothermophilus strain 10 was obtained from L. L. Campbell (University of Delaware, Newark). Thermus sp. X-l(14) and Thermomicrobium roseum ATCC 27502 were isolated previously i n this laboratory (10). Media and growth conditions were similar to those used for the characterization of Thermus sp. X-1 (14) and Thermomicrobium roseum (10).
Absorbancy spectrum. The absorbancy spectrum
of the acetone extract of isolate K-2 was determined
with a Cary 15 recording spectrophotometer. The
acetone extract was made from exponentially growing cells t h a t had been centrifuged, suspended in
0.05 M potassium phosphate (pH 8.0), centrifuged
again, suspended in acetone with a Ten-Broeck homogenizer, and centrifuged; the supernatant was
used for the absorbancy spectrum. A duplicate sample was placed on a predried, preweighed planchet,
dried a t 90 C, and used to determine the total dry
weight.
Phase-contrast photomicrography. A Carl Zeiss
RA phase microscope with a x 100 Neofluor objective
was used for all microscopy observation. Photomicrography was performed with a Zeiss camera with
Kodak Monochrome film (SO-410 ESTAR-AH base
135-36; Eastman Kodak Co., Rochester, N.Y.).
Scanning electron microscopy. A segment of
agar carrying a single 48-h-old colony of the K-2
isolate was cut out and fixed in modified Zamboni
fixative (16) for several hours. The Zamboni fixative
was modified by dilution to 50% (vol/vol) with water
and addition of glutaraldehyde to a final concentration of 1% (vol/vol). A smear from a similar colony
was made on a glass cover slip and fixed in the same
manner. Postfixation was carried out in 1% osmium
tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h.
The osmium was then cross-linked by treating the
specimens with thiocarbohydrazide (saturated solution in water) for 20 min (11) and treating them
again with osmium for 1 h. This treatment did not
completely prevent electrostatic charging in the
scanning electron microscope, so the specimens were
subsequently coated with successive thin layers of
carbon and gold in a vacuum evaporator. The speci-
357
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358
INT.J. SYST.BACTERIOL.
RAMALEY ET AL.
mens were viewed in a scanning electron microscope
at 20 kV. The original micrographs were taken on
Polaroid 55 PN film.
DNA base ratio. Deoxyribonucleic acid (DNA)
was isolated by the method of Marmur (12). Treatment with lysozyme and complete homogenization
of the bacteria after addition of the detergent and
phenol with a Ten-Broeck homogenizer were necessary to effect complete release of the DNA from this
and other similar gram-negative, thermophilic bacteria. The buoyant density of the different K-2-type
DNAs in cesium chloride was determined by density
gradient ultracentrifugation as reported previously
with Escherichia coli bacteriophage T2 as reference
DNA (14).
Chemicals. Yeast extract, tryptone, and agar
were obtained from Difco Laboratories, Detroit,
Mich. The biochemicals were obtained from the
Sigma Chemical Co., St. Louis, Mo. All other chemicals were from the Fisher Scientific Co. Pittsburgh,
Pa. All water used was tap-distilled water which
passed through a mixed-bed ion-exchange resin. In
some cases, doubly glass-distilled water was used,
but no significant difference in results was observed.
RESULTS
Isolation of the K-2 isolate. The K-2 isolate
was isolated in 1972 from thermally polluted
streams on the Indiana University campus in
Bloomington, Ind. Isolates resembling the K-2
isolate were obtained from natural hot springs
in Colorado during the summer of 1973. The
area on the Indiana University campus that
yielded the highest number of K-2-type isolates
(30% of the total number of thermophilic bacteria) was in the lower portion of a thermal gradient that was caused by the constant discharge
of hot water (90C) from a pipe alongside a stone
bridge across the Jordan River north of Jordan
Avenue. This area is close to station 30b previously investigated by Brock and Yoder (4). The
Colorado hot spring thermal gradients that
yielded the highest numbers of K-2-type isolates were a t Poncha Hot Springs (7, 18) (Poncha Spring, Colo.) and Routt Hot Springs (7,181
(7 miles [ca. 11.3 km] north of Steamboat
Springs). Neither of these springs has detectable levels of sulfide (7). Other springs, such as
Waunita Hot Springs east of Gunnison, Colo.,
that have a higher level of sulfide (7, 18)did not
yield any of the K-2-type isolates.
It is interesting that the K-2-type isolate is
most often found in partially shaded thermal
gradients in which the bacterial mat has been
disturbed and is regrowing, and not in highlight areas such as the eMuent channels of
slightly alkaline hot springs at Yellowstone National Park.
Properties of the K-2 and K-2-type isolates. (i) Optimum growth temperature. All
of the isolates obtained to date have an optimum growth temperature of 60 C (Fig. 1)when
grown in 0.1% (wt/vol) yeast extract-0.1%
(wthol) tryptone in Castenholz salts at pH 7.6.
The K-2 isolate only grows in a slightly alkaline medium (pH optimum of 7.6 to 7.8). In
static tube cultures of the K-2 isolate, visible
growth on the surface of the liquid medium can
be observed after 2 weeks at 80 C, the apparent
upper temperature limit of growth for the K-2
isolate.
(ii) Absorption spectra of the K-2 and K2-type isolates. Figure 2 shows the absorption
spectrum of an acetone extract of the K-2 isolate. All of the K-%type isolates examined have
7-
30
50
40
60
Temperature, c"
80
70
FIG. 1. Effect of temperature on the growth rate of
the K-2 isolate.
'3)50
4O:
4;O
S;O
Wauelenilh Inm)
5;O
-
8
O
;
650
100
FIG. 2. Absorption spectrum for the acetone extract of the K-2 isolate. The acetone extract (from 50
mg-eq dry weight of cells per ml of acetone) was
prepared from exponentially growing cells o f the K-2
isolate.
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VOL. 25, 1975
NEW, THERMOPHILIC GRAM-NEGATIVE BACTERIUM
the same pink-to-light red colonial pigmentation and absorption spectrum. The absorption
spectrum is quite distinct from that of thepalepink, pleomorphic, thermophilic bacterium
Thermomicrobium roseum isolated from Yellowstone National Park (10). The pink pigmentation of the K-2 isolate is not water soluble and
is bound firmly to the isolated cell membrane.
This pigmentation is presumably due to carotenoids, which are currently being investigated
by J. J. Cooney of the University of Dayton,
Dayton, Ohio. There is no present evidence
that light is required for pigment production.
(iii) DNA base ratios of the K-2 and K-2type isolates. Table 1 shows the DNA base
ratios of the K-2 isolate (Bloomington, Ind.),
the P-P-1 isolate (Poncha Hot Springs, Colo.),
and the R-P-1 and R-P-2 isolates (Routt Hot
Springs, Colo.). The guanine plus cytosine
(G+C) contents of the DNAs of all of the isolates are similar, with an average value of 64.4
2 0.6 mol%, as determined by E. Carusi of the
Creighton Medical School in Omaha, Neb.
Thus, the DNA base ratios of the K-2 and K-2type isolates are very similar to those of other
gram-negative thermophilic bacteria found in
slightly alkaline thermal aquatic environments, e.g., Thermus X-1 (64 to 65 mol% G+C)
(13; unpublished data), Thermus aquaticus
(65.4 to 67.4 mol% G+C) (31, and Thermomicrobium (64.3 mol% G+C) (10).
(iv) Morphology of the K-2 and K-%type
isolates. The isolates all have a similar colonial and cellular morphology. Figure 3 shows a
phase-contrast photomicrograph of the R-P-1
isolate from Routt Hot Springs, and Fig. 4A
shows a scanning electron micrograph of the
peripheral surface of a colony of the original K2 isolate. Figure 4B shows a scanning electron
micrograph of some individual cells of the K-2
isolate on the agar surface, and Fig. 4C shows
some single cells smeared onto a cover slip. The
TABLE1. DNA base ratios
Strain no.
Source
I
359
fixative method used for these cells produced
very little shrinkage.
Repeated measurement of the different isolates under phase-contrast observation with an
ocular micrometer calibrated with a stage micrometer gave a n average cell diameter for the
K-2 isolate of between 0.7 and 0.8 pm and an
average length of between 5 and 8 pm. Similar
measurements of a Thermus isolate give a
slightly smaller dameter (0.6 to 0.8 pm) and
filamentous forms up to 100 pm (3) in length,
depending on cultural conditions and the temperature employed.
(v) Growth requirements of the K-2 isolate.
Table 2 lists some of the media tested that
supported the growth of the K-2 isolate in static
culture. A number of different complex carbon
sources a t low concentration can serve for the
growth of the K-2 isolate. Although some
growth can be obtained with glutamate as the
sole carbon source and with other simple minimal media in liquid culture, the K-2 isolate will
not grow upon repeated transfers on glutamate
agar, suggesting additional growth factor requirements beyond those of Thermus. The K-2
isolate showed no nitrate reductase activity
and, like Thermus aquaticus, cannot use nitrate as a significant nitrogen source. In contrast, Thermus X-1 has a very active nitrate
reductase (R. Ramaley and R. Carroll, unpublished data).
DISCUSSION
Table 3 presents a summary of some of the
properties of the K-2 isolate in comparison with
other thermophilic bacteria. In the practical
identification of the K-2 isolate, its morphology, obligate thermophily, and characteristic
pink-to-red pigmentation make it quite easy to
identify. However, its taxonomic relationship
with the other thermophilic, gram-negative isoof
the K-2 and K-2-type isolates
DNA bouyant
density (g/cm3)
MolPloG+C,,
Ultracentrifugation
performed by
K-2
Bloomington, Ind.
1.722
63.8
L. P r a t h e r
K-2
Bloomington, Ind.
1.723
64.3
E. Carusi
P-P- 1
Poncha Hot Springs, Colo.
1.723
64.3
E. Carusi
R-P-1
Routt Hot Springs, Colo.
1.724
65.3
E. Carusi
R-P-2
Routt Hot Springs, Colo.
1.722
63.8
E. Carusi
The average a n d standard deviation for all the G + C values shown is 64.3
standard deviation for t h e last four determinations is 64.4 ? 0.6 mol%.
2
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0.6 mol%. The average a n d
360
INT. J. SYST.BACTERIOL.
RAMALEY ET AL.
FIG. 3. Phase-contrast photomicrographs of the isolate from Routt Hot Springs (R-P-1). Marker bar
indicates 10 pm.
lates (Thermus, etc.) that are also found in
slightly alkaline thermal environments is quite
unclear. One possibility would be to create a
new genus for the K-2 isolate, as was done for
Thermus aquaticus (3), Thermomicrobium
(lo), Sulfolobus (Z), and Thermoplasma (6).
This is, perhaps, the least satisfactory solution
considering the continuing isolation of physiologically similar thermophilic gram-negative
isolates and the eventual confusion that would
result from creating an excessive number of
genera for them. Premature assignment as to
genus and species have already confused the
thermophilic isolates of Heinen now that it has
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VOL. 25, 1975
NEW, THERMOPHILIC GRAM-NEGATIVE BACTERIUM
been shown that these isolates are sporeforming bacteria (8, 9).
A second possibility would be t o place K-2 in
a separate species in an existing genus such as
Thermus. The base ratio of the K-2 DNA falls
within the range designated for Thermus. Like
Thermus strains, it only grows on dilute organic media, is an obligate aerobe, and produces only acid from carbohydrates. It resembles Thermus strains morphologically although
it does not seem to form the characteristic long,
filamentous cells. However, there are some yellow Thermus isolates that have very transulent, spreading colonies and that do not seem to
form the long filaments (13; unpublished data)
of the type species Thermus aquaticus.
According t o the diagnostic keys in the 8th
edition of Bergey’s Manual of Determinative
Bacteriology (51, the K-2 isolate keys out to
Thermus, except for its optimum growth temperature (60 versus 70 to 72 C for Thermus)and
its pigmentation (pink versus nonpigmented to
yellow-orange for Thermus).It is entirely possible that the K-2 isolate is a member of a species
of Thermus. However, to place it in a new
species of Thermus would also require a broad-
361
ening of the description of Thermus with respect to optimum temperature and pigmentation.
Another alternative would be to place the K2 isolate in one of the species of Flauobacterium
with a high G + C value since “. . . one of the
functions of this genus has always been to be
used rather nondiscriminantly as a category for
pigmented, aerobic rods that are usually gramnegative and anaerogenic in carbohydrate-containing medium. This use of Flauobacterium as
a category of taxonomic convenience is precisely what the originators of the genus intended” (0. B. Weeks, personal communication).
The K-2 isolate keys into the genusFlauobacterium as a second possibility, and at least one
red-pigmented marine bacterium has been assigned to Flauobacterium ( 5 ) . However, Oshima and Imahori (13) removed a thermophilic
organism from the genus Flauobacterium and
placed it in Thermus.
Since the K-2 isolate and other gram-negative, thermophilic bacteria (Thermus strains,
etc.) are being used increasingly in this and
other laboratories (3) for comparative biochemi-
FIG.4. Scanning-electron photomicrographs of the K-2 isolate. (A) Surface of a young (48-h-old)colony;
(B>isolated cells on the agar surface; (C) isolated cells i n a smear made o n a glass cover slip. T h e marker bar
indicates 1 p m .
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362
INT. J. SYST.BACTERIOL.
RAMALEY ET AL.
FIG. 4B and C
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363
NEW, THERMOPHILIC GRAM-NEGATIVE BACTERIUM
VOL. 25, 1975
cal studies, it is important that the different
isolates be characterized and identified so far as
possible. The K-2 isolate appears to belong to a
new species, but assignment to any of the curTABLE2 . Media supporting growth of the K-2
isolatea
Compounds
(0.1%, wtlvol)
added to
basal salts
media
Growth i n basal
salts media
Castenholz
(nitrate)
Allen (10)
(ammonia)
++
++
+
+-
++
++
+++
-
-
+
+
+
+-
++
Yeast extract plus tryptone
Yeast extract
Tryptone
Peptone
Casein hydrolysate
Tryticase soy
Nutrient broth
Mannitol
Glutamate
++
+
-
+ + , Heavy growth; + , light t o moderate growth; + -,
trace; -, no growth. No growth with fructose, glucose,
glycerol, succinate, acetate, or citrate under the conditions
tested after 1 week. All media had a n initial pH of 7.8.
TABLE3 . Summary
of
Property
rently recognized genera is difficult. Thus,
pending a revision of the genus Thermus as
new Thermus isolates are obtained (5), the K-2
isolate is referred to simply as a pink, obligately thermophilic, unidentified bacterium. A
culture of this strain has been deposited in the
American Type Culture Collection under the
number 27599. Studies of the comparative properties of some of the enzymes and other cellular
components of the K-2 isolate and of the ecological distribution of the K-2 isolate will be published elsewhere.
ACKNOWLEDGMENTS
This work was supported by National Science Foundation grant GB-36931. The initial isolation and preliminary
characterization of the K-2 isolates were made while the
three senior authors were associated with the Department
of Microbiology at Indiana University, Bloomington. The
studies of the Colorado hot springs were conducted from the
Rocky Mountain Biological Laboratory at Crested Butte,
Colo., with the assistance of Alan Ramaley. We would like
to thank E. Carusi (Creighton Medical School, Omaha,
Neb.) for th e determination of DNA base ratios and Linda
Malick (Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha) for th e processing
of samples for the scanning electron microscope.
REPRINT REQUESTS
Address reprint request to: Dr. Robert F. Ramaley, Department of Biochemistry, University of Nebraska, College
of Medicine, Omaha, Neb. 68105.
LITERATURE CITED
1. Brock, T. D. 1970. High temperature systems. Annu.
the properties of the K-2 isolate and other thermophilic bacteria found in slightly
alkaline thermal environments
K-2 isolate
Thermomicrobium roseum
Thermus
Thermus
x-l
YT-1
Bacillus stearothermophilus 10
Colony color
Pink
Pale pink
Yellow
Cream
Cream to brown
Optimum growth temp"
(C)
60
70-75
70
70
60-65
Generation time" (min)
50
360
60-70
50-60
12
pH range for growth"
7.5-10
8.0-10
7.5-9.0
7.5-10
5.0-9.0
Cell morphology
Thin rods
Short, pleomorphic
rods
Thin rods
Thin rods
Sporeforming, wider
rods
Growth on standard nutrient agald
No
No
No
No
Yes
Growth
agaP
glutamate
No
Yes
Yes
Yes
Yes
Production of nitrite from
nitrate
No
No
No'
Yes
Yes
on
~~
~~~~
~
~
~~
~~
Determined from turbidity increase a s described in t h e text.
* Production of single, isolated colonies away from initial streak. Some of th e original medium was usually carried over in
the initial streak area and, combined with the high number of organisms, may have led to apparent growth.
' Very low level of nitrite believed not to be significant.
It
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364
INT. J. SYST.BACTERIOL.
RAMALEY ET AL.
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